AES-I is a highly transmissible virulent strain of P. aeruginosa
that has been identified in several CF clinics along the eastern seaboard of Australia where surveillance programs have been introduced [1
]. We have previously shown that patient segregation has successfully limited the spread of AES-I in a CF clinic in Melbourne [14
]. There is currently an urgent need for a diagnostic tool that will enable rapid identification of CF individuals that harbour the AES-I strain so that appropriate segregation measures can be employed during hospital and clinic visits. Toward this end, we have used SSH and PCR to identify 2 genetic loci (HW2 and HW3) that are highly conserved amongst AES-I isolates of P. aeruginosa
. PCR was utilised to profile the presence of these AES-I loci in a total of 188 P. aeruginosa
strains. These screens found that the HW3 locus was absent in all 153 non-AES-I isolates but present in only 30/35 of the AES-I strains (86% sensitive and 100% specific) indicating that this locus is present in a region of some variability between AES-I isolates. The HW2 locus was found to be present in all 35 strains determined by PFGE to be AES-I and absent in all 153 non AES-I P. aeruginosa
strains tested. These results indicate that PCR for the HW2 locus is 100% specific and 100% sensitive for detection of the AES-I epidemic P. aeruginosa
The collection of strains used in this study included isolates from CF individuals that were identified in our initial 1999 clinic surveillance to harbour the AES-I strain [9
] as well as isolates obtained over the subsequent 6 years from the same CF individuals. The HW2 locus was present in all initial and subsequent AES-I isolates obtained from the same individual indicating that this locus did not display variability during chronic infection over this time period.
The main purpose of the oprL
PCR assay in our study was to act as a positive control for DNA quality to avoid false negative results in our PCR screens. Since we commenced our clinical surveillance study in 2005, it has been shown that the ecfX
locus is more specific than oprL
to identify P. aeruginosa
from various environmental and clinical extracts [30
]. Accordingly, a PCR assay for the ecfX
locus could be substituted for the oprL
assay to provide greater specificity for the presence of P. aeruginosa
in sputum samples if desired.
Compared with PFGE which can take 6 to 8 days to obtain a diagnosis from time of receipt of patient sputum, PCR-based assays have many benefits as potential diagnostic tools as they are simple, relatively inexpensive, do not require highly skilled personnel, reduce handling and detection errors and can significantly reduce the time required to identify patients harbouring the AES-I strain. We have shown that PCR amplification of the oprL and HW2 loci can be used for the direct detection of P. aeruginosa and AES-I in CF sputum swabbed onto Whatman FTA® Elute cards. We are now further assessing the potential for these assays for use as routine surveillance tools for P. aeruginosa and AES-I in CF sputum. The use of Whatman FTA® Elute cards for sputum storage, transport and DNA template purification for PCR is simple and relatively inexpensive and could be easily adapted for detection of other bacterial pathogens from a broad range of respiratory and other infections.